Molecular mechanisms underlying formin-associated inherited deafness Summary This R15 AREA proposal will support investigations of the molecular mechanisms by which cytoskeletal structures in the inner ear are built and maintained. Mutations of the human Diaphanous 1 (DIAPH1) gene causes autosomal dominant, non-syndromic, progressive hearing loss, referred to as DFNA1. The molecular mechanisms underlying this form of deafness are unknown. DFNA1 could serve as a model for inner ear maintenance over a lifetime. DIAPH1 is a member of the formin family of actin and microtubule cytoskeletal regulators. The inner ear contains several precisely constructed cytoskeletal structures, most notably the bundle of stereocilia on hair cells. Investigations of deafness-associated cytoskeletal regulators will contribute to our understanding of biology of the inner ear. This project will use DFNA1 mutations as a starting point for understanding the role of formins in the inner ear. The proposed work will test three related hypotheses: (1) that the deafness-associated mutations in DFNA1 disrupt the protein's intramolecular regulatory interaction, (2) that the mutations compromise the ability of DIAPH1 to regulate the assembly of actin filaments, and (3) that the same mutant proteins are compromised in their ability to collaborate with other actin regulatory proteins that are highly expressed in the hair cells of the inner ear. All three hypotheses are plausible given that the locations of several DFNA1 mutations coincide with a domain that interacts with cytoskeletal filaments, intramolecular regulatory domains, and other cytoskeletal regulatory proteins. To test these hypotheses, a set of reductionist biochemistry experiments are proposed, including protein:protein interaction and actin filament assembly assays. These experiments are accessible for undergraduate researchers, and the overarching research questions will be of interest to students pursuing careers in the biomedical sciences.